Osteocalcin is the second most abundant protein in bone, yet little is known about its function. It appears at the time of mineralization, although it is not clear whether this is a temporal or functional relationship. Osteocalcin has also been implicated in the recruitment of osteoclasts, and measurements of serum osteocalcin levels have been used as a clinical index of bone turnover. Understanding the role of this protein in bone is expected to increase our knowledge of the regulation of bone mineralization and remodeling, and may broaden the usefulness of serum osteocalcin determinations in the clinical setting. To elucidate the function of osteocalcin, a line of mice with targeted disruption of the osteocalcin gene will be engineered. Such mice will be unable to synthesize osteocalcin. Analyses of defects in skeletal development and bone remodeling in these mice are expected to define the role of this protein in the vertebrate skeleton. The induction of osteocalcin gene transcription by 1,25-dihydroxyvitamin D3 has helped to characterize the interactions of the 1,25-dihydroxyvitamin D3 receptor with target DNA sequences that mediate transcriptional induction by 1,25-dihydroxyvitamin D3. It is anticipated that, in much the same manner, investigations of the developmental and tissue-specific regulation of osteocalcin gene transcription will help to define the determinants of the osteoblast phenotype. Osteocalcin is expressed only in mature osteoblasts, hence, the osteocalcin gene will also serve as a model for unravelling the complex series of events involved in the differentiation of pluripotential cells into osteoblasts. Stromal cells will be treated with Bone Morphogenic Protein-2 to induce their differentiation into osteoblasts, which express the endogenous osteocalcin gene. Transfection of osteocalcin-CAT fusion genes concomitant with BMP-2 treatment will identify DNA sequences that are responsible for the developmental expression of the osteocalcin gene. Factors that interact with these DNA sequences to induce osteocalcin gene transcription in these stromal cells will be identified. Characterization of these factors and investigations of their regulation and activation will help to characterize the series of molecular events that occur between BMP-2 treatment and the acquisition of the osteoblast phenotype We have identified sequences in the rat osteocalcin gene that contribute to the silencing of osteocalcin-CAT fusion genes in cell lines that do not express the endogenous osteocalcin gene. Factors that interact with these DNA sequences to confer tissue-specific expression will be identified. Understanding the regulation of these factors, and how they interact with the osteocalcin gene will help to define the phenotype of the mature osteoblast.